Functional Characterization of Novel Tautomycetin Analogues through tmcK Gene Disruption in Streptomyces CK4412

초록

Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is an antifungal secondary metabolite with an unusual ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Recently, TMC was identified to possess additional biological activities such as T cell-specific immunosuppressive and colorectal anti-cancer activities through probable inhibition of protein phosphatase activities, leading us to isolate and characterize its entire biosynthetic and regulatory pathway gene cluster. To understand tailoring mechanism of TMC biosynthesis as well as to provide novel lead compounds for potential chemotherapy agents, we generated and characterized several mutant strains containing inactive tailoring enzymes via specific targeted gene disruption. Among genes encoding these tailoring enzymes, tmcK with a deduced product of 485 amino acid residues located on the 3‟-terminus of polyketide synthase genes (tmcAB), was found to have amino acid sequence homology with putative bacterial decarboxylase genes. Inactivation of tmcK from the Streptomyces sp. CK4412 chromosome resulted in a 5-deoxy-3”-carboxylic TMC analogue, whose structure was confirmed using LC-MS and NMR analyses. Moreover, this tmcK mutant strain was functionally complemented by an integrative plasmid carrying tmcK to restore TMC biosynthesis, suggesting that TmcK is indeed a functional polyketide decarboxylase involved in TMC biosynthesis. The fact that a 5-deoxy-3”-carboxylic TMC in tmcK mutant showing significantly different biological and pharmacokinetic activities implies the possibility of producing rationally-redesigned TMC derivatives using targeted gene modification.